1. EVALUATION OF CYTOTOXIC EFFECT OF
PHYTOCONSTITUENTS OF TINOSPORA
CORDIFOLIA USING 3T3L1 FIBROBLAST
CELL LINE
PRESENTED BY
PRIYANKA CHAURASIA

2. THE STUDY…….

prevalence of genetic and stress related diseases is
on increasing trend such as diabetes.

Tinospora cordifolia - antidiabetic activity.

3T3L1 cell line - pivotal in understanding - basic
cellular mechanisms associated with diabetes,
obesity and related disorders.

present study - to evaluate the cytotoxic potential of
the Tinospora cordifolia and its isolated fractions in
3T3L1 cells using an MTT assay.

3. INTRODUCTION

Diabetes mellitus - global health crisis - attained a
pandemic form.

important to control diabetes and its complications alleviate the human suffering.

Prior to investigating the plants for their anti-diabetic
activity, it is first essential to evaluate their cytotoxic
potential.

Evaluating in vitro cytotoxic potential - provide important
preliminary data - help select plant extracts
with potential anti-diabetic potential for future work.

4. 
Tinospora cordifolia used for the treatment of
diabetes.

Oral administration of an aqueous T. cordifolia root
extract (TCRE) to alloxan diabetic rats caused a
significant reduction in blood glucose and brain
lipids, increase in body weight, total haemoglobin &
hepatic hexokinase.

lowers hepatic glucose-6-phosphatase and serum
acid phosphatase, alkaline phosphatase, and lactate
dehydrogenase in diabetic rats.

hypoglycaemic and hypolipidaemic effect.

5. AIMS AND OBJECTIVES

Aim:
To evaluate the cytotoxic potential of Tinospora
cordifolia & its isolated fractions using 3T3L1
Fibroblast cell line.

Fractions of T. cordifolia:

isolated fractions viz., Water, Butanol, Undissolved
and Ethyl acetate fractions using MTT assay.

6. TYPE 2 DIABETIC
COMPLICATIONS
1. HEART AND BLOOD VESSEL DISEASE : Increases the risk of various cardiovascular problems.
 risk of stroke and death rate is more.
2. NEUROPATHY(NERVE DAMAGE) : Nerves damage - problems with nausea, vomiting,
diarrhoea or constipation.
3. NEPHROPATHY(KIDNEY DAMAGE) : Severe damage - kidney failure or irreversible end-stage
kidney disease, requiring dialysis or a kidney transplant.
4. EYE DAMAGE : Retina damage – blindness – condition termed as
Diabetic Retinopathy
 Serious vision conditions – cataracts & glaucoma

7. 3T3L1 CELL
LINE


3T3-L1 is a cell line derived from 3T3- Swiss albino
mouse cells that is used in biological research on
adipose tissue.
Source – Adipose tissue

3T3-L1 cells have a fibroblast-like morphology and cells
differentiate into adipocyte phenotype.

3T3L1 cells of adipocytes increases the synthesis and
accumulation of triglycerides.

3T3 cell line established by two scientists, George
Todaro and Howard Green in 1962.

8. 3T3L1 CELLS
Fibroblast phenotype
Adipocyte phenotype

9. OTHER CELL LINES THAT CAN BE
USED FOR IN VITRO STUDIES ON
DIABETES
CELL LINE
CELL ORIGIN
SPECIES
MIN 6
INSULINOMA
MOUSE
RINm
INSULINOMA
INSULINOMA
RAT
DISADVANTAG
ES
EXPRESS
GLUCOKINAS
E & GLUT 2
TREATMENT
WITH
NICOTINAMIDE
MAKES THEM
RESPONSIVE
TO GLUCOSE
RAT
INS 1
ADVANTAGE
S
INSULIN LEVEL
DECREASES.
SECRETES
SOMATOSTATIN
.
NOT RESPOND
TO GLUCOSE
RESPONDS
TO
GLUCOSE.
REQUIRE
MERCAPTOETHANOL IN

10. KNOWN ANTI-DIABETIC
DRUGS

Modern Drug:
Sulphonylureas, Pioglitazone, Metformin, Thiazolidinediones etc.


Pioglitazone is known insulin sensitizer.
Ayurveda drugs:
Phyllanthus emblica, Allium sativum, Aloe vera, Tinospora
cordifolia.

Tinospora cordifolia is widely used for its antidibetic properties.

11. TINOSPORA CORDIFOLIA

Tinospora cordifolia (Guduchi) is a large, glabrous,
deciduous climbing shrub belonging to the family
Menispermaceae.

Chemical constituents from this shrub belong to different
classes - alkaloids, diterpenoid lactones, glycosides,
steroids, sesquiterpenoid, phenolics, aliphatic
compounds and polysaccharides.

medicinal properties - anti-diabetic, anti-periodic, antispasmodic, anti-inflammatory, anti-arthritic, antioxidant, anti-allergic, anti-stress, anti-leprotic, antimalarial, hepatoprotective, immunomodulatory and
anti-neoplastic

12. PHARMACOLOGICAL
ACTION

aqueous extract of guduchi stem has shown the presence
of arabinogalactan that showed immunological activity.

stem is used in dyspepsia, fevers and urinary diseases.

reduces oxidative stress and lowers blood sugar level in
body.

used in leprosy, diabetes, cardiac debility, jaundice and
general weakness.

used in inflammations, gout and skin diseases.

13. CYTOTOXIC EFFECT

Guduchi is very well established immunomodulatory
agent and proven antioxidant herb.

Dichloromethane extract is the most promising one as far
as cytotoxic effect is concerned. These preliminary studies
were done to establish the cytotoxic effect of guduchi at
higher doses.

The anti tumor activity of TC may be due to decreased lipid
peroxidation, glutathione-S-transferase activity or due to the
release of lactate dehydrogenase.

14. OTHER CYTOTOXICITY
ASSAYS
1. LDH assay:
The LDH assay give satisfactory responses by using cell
membrane damaging agents like triton X-100; e.g. sodium
azide which inhibits the respiratory chain.
2. Neutral red assay:
This assay is less sensitive (excitoxic model) & cannot
recommend it for the use in ion channel studies.
Measures the cell death.
3. ATP based assay:
It is the most sensitive assay. The disadvantage of this
method is the luminescence-readout, which could be
influenced by quenching side effects in the samples.

15. OUTLINE OF PROJECT
TC
SOLUBILIT
Y
D/W
DMS
O
VIABILITY
MTT
ASSAY

16. FRACTIONS OF TC

Water- soluble in 100% D/W.

Butanol- soluble in 100% D/W.

Undissolved- soluble in 100% D/W.

Ethyl acetate- soluble in 100% DMSO when kept
overnight.

17. PRINCIPLE OF MTT ASSAY

This is a colorimetric assay that measures the reduction of
yellow 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium
bromide (MTT) by mitochondrial succinate
dehydrogenase.

The MTT enters the cells and passes into the mitochondria
where it is reduced to an insoluble, coloured (dark purple)
formazan product.

Since reduction of MTT can only occur in metabolically
active cells the level of activity is a measure of the viability
of the cells.

18. RESULTS
Aqueous extract of
Tinospora cordifolia:
Water fraction of
Tinospora cordifolia:
0.5
0.45
1
0.8
0.35
Absorbance at 570 nm
Absorbance at 570 nm
0.4
0.3
0.25
0.2
0.15
0.1
0.6
0.4
0.2
0.05
0
Control
cells
1
5
10
50
0
Control cells
1
5
10
50

19. Butanol fraction of
Tinospora cordifolia:
Undissolved fraction of
Tinospora cordifolia:
1
1
0.8
Absorbance at 570 nm
Absorbance at 570 nm
0.8
0.6
0.4
0.2
0.6
0.4
0.2
0
0
Control
cells
1
5
10
50
Control
cells
1
5
10
50

20. Ethyl acetate fraction of Tinospora
cordifolia
0.7
Absorbance at 570 nm
0.6
0.5
0.4
0.3
0.2
0.1
0
Control cells
1
5
10
50

21. DISSCUSSION

Aqueous extract of Tinospora cordifolia and its 5 fractions
viz., Water, Butanol, undissolved and Ethyl acetate fraction
were studied over a concentration range of 1 to 50 µg/ml.

aqueous extract of Tinospora cordifolia, Water, Butanol
and Undissolved fraction - not show a significant
increase in the absorbance values for a time period of 24
hours.

ethyl acetate fraction - a dose dependent decrease in
the absorbance as compared to untreated cells.

necessary to study EAF at lower concentrations below 5
μg/ml to evaluate its cytotoxic potential.

22. CONCLUSION

The present study was conducted to evaluate the cytotoxic
potential of aqueous extract of Tinospora cordifolia and its
isolated fraction.

observed - aqueous extract, Water, Butanol and
Undissolved fraction - not affect the viability of the 3T3L1
cells at the concentrations studied - no cytotoxic effect.

Ethyl acetate fraction of Tinospora cordifolia - a
concentration dependent effect on the viability of the
3T3L1 cells - cytotoxic effect.

All the fractions except ethyl acetate can be further
evaluated for their anti diabetic potential

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
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
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
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
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
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
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
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